There is growing interest in the study of RNA modifications and its potential role(s) in many physiological abnormalities, such as modulating many processes associated with cancer development and progression. While RNA sequencing (RNA-seq) using short-read nextgeneration sequencing (NGS) have proven to be a useful tool for identifying specific RNA modifications, there still remains technical challenges. These technical challenges include: [1] a harsh bisulfite conversion generating RNA fragments followed by a required purification step; [2] an intermediate cDNA synthesis protocol to incorporate necessary 3? adaptors followed by a required purification step; [3] a PCR protocol (to incorporate 5? library barcodes) to create a heterogeneous pool of sample library templates for NGS sequencing that may require optimization while promoting potential sampling bias and still another required purification step; and [4] a quality control step to verify library enrichment with correct base-pair size distributions prior to sequencing. Importantly, using any of these pre-analytical steps, important RNA modification information can be lost. Exonuclease Time-of-Flight (X-TOF) is a transformative single-molecule sequencing technology specifically aimed at addressing each of these challenges by employing a sequencing-bysubtraction approach for the detection of methylation status (or other types of modifications) imposed on single molecules (ribonucleotide monophosphates, rNMPs) generated sequentially from full-length native RNA species. The workflow is intended to be simple requiring only purified RNA as input without the need for bisulfite conversion, eliminating the need for cDNA synthesis and PCR, and incorporates an internal quality control RNA to verify system performance. Compared to existing single-molecule sequencing strategies, such as the Oxford Nanopore?s MinION, X-TOF provides the following unique attributes: [1] Uses multiple singlemolecule identifiers (time-of-flight, TOF; current transient amplitudes) to identify nucleotides with unprecedented accuracy - >95%; [2] can process input samples, RNA, <1 ng negating the need for a PCR amplification step to sequence rare targets; [3] because X-TOF is a chip-based technology, it can be easily integrated to microscale sample processing units to provide fully automated processing ? bona fide sample-to-sequencing platform; and [4] X-TOF chip is madein a plastic to allow for high-scale production at low-cost using injection molding, in spite of the fact that the platform has nanometer structures.